Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr–/– and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.


Supplemental Table 1
Table S1.List of primers used for gene expression analysis.

Supplemental Figure S2
Characteristics of advanced plaques in aged mice.Representative micrographs and quantifications of histological analysis from the aortic root of aged (68-73 weeks old) male LDLr -/-mice (n=14-13), treated with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control for 6 weeks while remaining on a chow diet, stained with (A) Oil Red O, (B) Masson's trichrome, (C) a fluorescent staining for macrophages (CD68, green), smooth muscle cells (α-SMA, red) and nuclei (DAPI, blue).Plaque tissue is outlined with a dashed line, necrotic core area is outlined with a dotted line in the Masson's trichrome micrographs.Quantifications expressed as mean ± SEM, unpaired T-test, no significant results, scale bar is 100 µm.

Supplemental figure S3
LMP7 inhibition directly inhibits DC activation and causes upregulation of constitutive proteasomal active subunits.(A) Bone marrow derived DCs derived from 8 week old male LDLr -/- mice were incubated with ONX-0914 (0 or 200 nM) overnight in absence or presence of LPS (100 ng/ml) and assessed by flow cytometry.(B) Cell culture viability was assessed by gating as shown in panel 'A'.For quantification of DC viability single cells were gated like in panel 'A', but thereafter CD11c + MHC-II + cells were gated followed by the gating of live DCs.CD86 median fluorescent intensity of the live MHC-II + CD11c + population was assessed as a measure for DC activation.(C) Bone marrow derived DCs were incubated with ONX-0914 (0, 50 or 100 nM) overnight in absence or presence of LPS (100 ng/ml) after which DC viability was determined by flow cytometry like explained in panel 'B', and (D) expression of catalytic constitutive and immuno-proteasomal subunits, and immunoproteasomal activators were determined by qPCR.Expressed as mean ± SEM, one-way ANOVA with Holm-Sidak posttest, *p < 0.05, **p < 0.01, *** p < 0.001 **** p < 0.0001.

Supplemental Figure S4
Tregs are not increased by ONX-0914 treatment in early and advanced atherosclerosis.(A) CD4 + T-cell FoxP3 + CD25 + and FoxP3 + CD25 -populations were determined with flow cytometry in the spleen blood, cervical lymph nodes, and mesenteric lymph nodes of female LDLr -/-(n=15) after treatment with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control treated for 7 weeks, while fed a WTD.(B) CD4 + FoxP3 + regulatory T cell levels in the spleen of aged mice (68-73 weeks of age prior to WTD feeding) fed a WTD for 6 weeks prior to sacrifice, during which mice were treated with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control, as determined by flow cytometry.(C) Female LDLr -/-mice (n=7) were fed a WTD for 27 weeks and treated with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control in the final week of WTD, after which mice were sacrificed and CD4 + FoxP3 + regulatory T cell levels in the spleen were determined with flow cytometry.Expressed as mean ± SEM, unpaired two tailed t-test, * p < 0.05.

Supplemental Figure S7
Supplemental Figure S8 Hepatic histology.(A) Male LDLr -/-mice (n=8) were fed a WTD for 4 weeks after which a baseline group was sacrificed.Remaining mice were treated for 7 weeks with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control treated (4% DMSO in PBS).Representative micrographs of liver sections from control and ONX-0914 treatment groups stained with Oil Red O (scale bar is 100 µm).

A
Glucose and insulin tolerance tests in WTD fed male LDLr -/-mice.Male LDLr -/-mice (n=6-7) were fed a WTD for 7 weeks after which mice were treated with ONX-0914 (10 mg/kg intraperitoneally, 3 times weekly) or control for 3 weeks while remaining on a WTD.(A) Thereafter, mice were fasted for 6h before blood glucose was assessed.Then mice (n=7 per group) (B) received an oral glucose bolus (0.06 g, 2 mg/kg for a mouse of 30 g), and blood glucose was measured 15, 30, 60, 90, and 120 minutes later, (C) or received an intraperitoneal insulin injection (0.03 Units, or 1U/kg for a 30 g mouse) and blood glucose was measured 15, 30, 60, and 90 minutes later.Expressed as mean ± SEM, * p < 0.05, a: Student's T-test, b,c: 2-way repeated measures ANOVA with Šídák's posthoc test.

Supplemental figure S9
Treatment with ONX-0914 does not lead to hepatotoxicity.Male LDLr -/-mice (n=8) were fed a WTD after which a baseline group was sacrificed.Thereafter, mice were treated for 7 weeks with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control treated (4% DMSO in PBS).(A) ALT and AST activity were assessed on blood plasma derived from blood collected at sacrifice after 7 weeks of control or ONX-0914 treatment.(B) Hepatic Cyp3A11 expression.Expressed as mean ± SEM, (A) 2-way repeated measures ANOVA with Sidak posttest, (B) two tailed t-test, no significant differences.

Supplemental figure S12
TG and TC clearance from the blood is not enhanced by ONX-0914 treatment.Female APOE*3-Leiden.CETP mice (n=8) were fed a WTD for 3 weeks after which they were treated with ONX-0914 (10 mg/kg, 3 times weekly, intraperitoneally) or control treated (4% DMSO in PBS) for 2.5 weeks.After 2.5 weeks mice received a final control or ONX-0914 injection and were fasted for 4h.

Supplemental Figure S15
Supplemental Figure S16 Immunoproteasomal inhibition does not cause intestinal inflammation.Small intestines of LDLr - /-mice fed a WTD for 28 weeks and treated with ONX-0914 or control for the final week, were assessed for (A) macrophage, (B) T cell, and (C) inflammatory cytokine content by gene expression analysis.Expressed as mean ± SEM, unpaired two tailed t-test, * p < 0.05.

Gating strategies
Gating strategy for various immune populations in the aortic arch (Figure 2A-D).CD45 + cells were gated, after which fixable viability dye low cells were gated to remove dead cells, and singlets were gated (used for cell count).Then, B cells (B220 + ) cells were gated.From the B220 -population CD11b high cells were gated from which classical monocytes (CD11b high Ly6C high MHC-II -), neutrophils (CD11b high Ly6C mid MHC-II -), atypical monocytes (CD11b high Ly6C low MHC-II -), and macrophages (CD11b high MHC-II + ) were gated.The macrophage population was then subdivided in a SSC high and SSC low population.From the B220 -CD11b low population dendritic cells were gated (MHC-II + CD11c + ).2E/F).CD45 + cells were gated, after which fixable viability dye low cells were gated to remove dead cells, and singlets were gated (used for cell count).Then Ly6C low B220 low cells were gated to gate the MCH-II + CD11c + dendritic cells.

Gating strategy for dendritic cells in mediastinal (heart) lymph nodes (Figure
Gating strategy for dendritic cells and macrophages (Figure 3).Cells were gated based on FSC-SSC, after which doublets were removed and fixable viability dye low cells were gated.Then MCH-II + CD11c + dendritic cells were gated, and subsequently F4/80 + red pulp macrophages were gated from the non-DC cell population in spleen samples.Gating shown for a representative spleen sample.

Gating of T cell analysis (Figure
Gating of myeloid cell populations in SVF from gWAT (Figure 6A).Cells were gated based on FSC SSC.From there, leukocytes were gated based on CD45 + expression.Thereafter live cells were gated (Fixable Viability Dye low ), and singlets were gated based on SSC-A and SSC-H.Gating shown on a representative SVF sample from the ONX-0914 treated group.6B).Singlets were gated based on FSC-H vs FSC-A.Subsequently, live cells were gated (Fixable Viability Dye low ).Thereafter highautofluorescent cells were gated out using an empty channel, leading to the plot depicted in Figure 6B, leukocytes were gated out based on CD45 + expression, and CD34 + cells were then gated.CD31 + cells were gated out to remove endothelial cells from the CD34 + population.Then adipose stem cells (SCA-I + CD105 + ) and preadipocytes were gated (SCA-I + CD105 -).Gating shown for a SVF sample from the control treated group.S4A).Fixed cells were gated based on FSC vs SSC.Subsequently, single cells were gated based on FSC-A and FSC-H, whereafter live cells were gated (Fixable Viability Dye low ).CD4 + cells were gated, after which FoxP3 + CD25 + and FoxP3 + CD25 -CD4 + Treg populations were gated.Gating shown for a representative spleen sample.

Gating of Neutrophils in SVF (Supplemental Figure S10C). Singlets were gated based FSC-A and
Thereafter, live cells (Fixable Viability Dye low ), low autofluorescent cells, and CD45 + cells were gated to gate the live single leukocytes.From there, Neutrophils were gated (Ly6G + ).Gating shown for a representative SVF sample from the control treated group.15 animals per group.Atherosclerotic lesion size is the most important parameter with the largest variability.A group size of 12-15 mice is usually adequate to observe a significant difference between groups when lesion averages differ more than 25% between groups.

Blinding
Histological analysis was performed on blinded samples.

Inclusion Criteria / Exclusion Criteria / Randomization
Healthy female LDLr -/-mice.Randomization based on age and weight after 27 weeks on a WTD.

Blinding
No blinding was performed.

In Vivo: Gastric Emptying
Study setup:

Sample Size
3 male LDLr -/-mice per group.Changes in gastric emptying (marked by GastroSence 750 signal) as study parameter.Setup as a small pilot experiment, the difference in GastroSense signal in the stomach between treatment groups was already significant with this sample size.

Inclusion Criteria / Exclusion Criteria / Randomization
Mice were all from the same litter, and were randomized on weight prior to WTD feeding (control; 28.7 ± 0.9 g, ONX-0914; 28.7 ± 0.5 g)

Blinding
No blinding was performed.In Vivo: Advanced atherosclerosis in aged LDLr -/-mice Study setup:

Sample Size
Changes in immune phenotype as main readout parameter.Because a higher variability in immune parameters and some dropouts due to the advanced age of the mice were anticipated, group sizes that are a little bit larger than we would typically use for the immunophenotyping of immunomodulatory interventions were used.

Blinding
Histological analysis was performed on blinded samples.

Groups
Figure S5 LMP7 inhibition does not impact splenic T-cell differentiation in early atherosclerosis.(A) Tcell proliferation as measured by thymidine incorporation of unstimulated or αCD3/αCD28 stimulated splenocyte cultures (n=7) derived from female LDLr -/-mice treated with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control treated for 7 weeks, while fed a WTD.(B) Cytokine levels in supernatant of the splenocyte cultures stimulated with αCD3/αCD28 overnight.(C) Overall CD4 + T-cell levels, (D) CD4 + Tem, Tcm and Th0 levels, and (E) overall CD8 + T-cell levels, and (F) CD8 + Tem, Tcm and Tc0 levels, in the spleen as determined by flow cytometry.Expressed as mean ± SEM, unpaired two tailed t-test.Supplemental Figure S6 .Memory and naïve T-cell effector populations in cervical lymph nodes.Quantification of flow cytometric analysis of (A) CD4 + T-cell content and (B) memory and naïve CD4 + T-cell populations, (C) CD8 + T-cells and (D) memory and naïve CD8 + T-cell populations in CLN of female LDLr -/- mice (n=15) fed a WTD for 7 weeks, during which mice were treated with ONX-0914 (10 mg/kg, 3 times weekly intraperitoneally) or control treated.Expressed as mean ± SEM, unpaired two tailed t-test, ****p < 0.0001.
(A) Plasma triglyceride levels and (B) cholesterol levels in plasma after 4h of fasting.(C) To determine clearance of TG and TC from the blood, VLDL like particles containing radiolabeled [ 14 C]cholesteryl oleate and glycerol tri[ 3 H]oleate were administered intravenously.Radioactivity in plasma was assessed in blood drawn at indicated time points after VLDL like particle administration, and half life time was approximated.Expressed as mean ± SEM (A,B) (left panel), C (1st and 3rd panel from the right), two tailed T-test, (B) (right panel) one-way ANOVA with Sidak posttest, C (1st and 3rd panel from the left) two-way repeated measures ANOVA with Sidak posttest, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Supplemental Figure S13 Depletion of peritoneal macrophages in gWAT.Female LDLr -/-mice (n=8) were fed a WTD for 27 weeks after which they were treated with ONX-0914 (10 mg/kg, 3 times weekly, intraperitoneally) or control treated (4% DMSO in PBS) for 1 week.A day prior to ONX-0914 treatment, half of the mice received clodronate liposomes or control liposomes.SVF from gWAT was isolated for flow cytometric analysis.(A) Representative flow cytometry plot of live single CD45 + cells gated for F480 + CD206 + resident macrophages and F480 + CD206 -infiltrating macrophages (B) Quantification of resident and infiltrating macrophage levels.(C) Flow cytometric gating of Ly6G + neutrophils in SVF, and (D) quantification of overall CD45 + immune cells and neutrophils in SVF of gWAT.Expressed as mean ± SEM, One-way ANOVA with Sidak posttest, * p < 0.05, ** p < 0.01.

Atherosclerosis initiation study Study setup: Sample Size:
WTD for 3 weeks.ONX-0914 treated (3 times weekly, intraperitoneally) for a week.Thereafter, oral administration of GastroSense 750 and cervical dislocation 30 minutes later.